Vault and vault-like carrier molecules

ABSTRACT

A method of using vaults as carrier molecules to deliver one or more than one substance to an organism, or to a specific tissue or to specific cells, or to an environmental medium. A vault-like particle. A method of preventing damage by one or more than one substance to an organism, to a specific tissue, to specific cells, or to an environmental medium, by sequestering the one or more than one substance within a vault-like particle. A method of delivering one or more than one substance or a sensor to an organism, to a specific tissue, to specific cells, or to an environmental medium. According to another embodiment of the present invention, there is provided a method of making vault-like particles, and making vault-like particles comprising one or more than one substance, or one or more than one sensor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing of International PatentApplication No. PCT/US04/07434 titled “Vault and Vault-Like CarrierMolecules,” filed Mar. 10, 2004 which claims the benefit of U.S.Provisional Patent Application No. 60/453,800 titled “Vault NanoCapsules” filed Mar. 10, 2003, the contents of which are incorporated inthis disclosure by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support underNational Science Foundation, Nano Science Interdisciplinary ResearchTeam Grant No. MCB-0210690 National Science Foundation Grant No.MCB-9722353. The United States Government has certain rights in thisinvention.

BACKGROUND

Vaults are ubiquitous, highly conserved cellular components found inphylogeny as diverse as mammals, avians, amphibians, the slime moldDictyostelium discoideum, and the protozoan Trypanosoma brucei. Scanningtransmission electron microscopic analysis has shown that the molecularmass of vaults is about 12.9±1 MDa, and cryo-electronmicrograph singleparticle reconstruction has determined that vaults have an overalldimension of about 420×420×750 Å. Thus, vaults have a greater mass andsize than many icosahedral viruses. The function of vaults is currentlyunknown.

Vaults are ribonucleoprotein particles comprising three differentproteins, designated MVP, VPARP and TEP1, and between one and threedifferent untranslated RNA molecules, designated vRNAs. For example, therat Rattus norvegicus has only one form of vRNA per vault, while humanshave three forms of vRNA per vault. The major vault protein, MVP, a 95.8kDa protein in Rattus norvegicus and a 99.3 kDa protein in humans, ispresent in 96 copies per vault and accounts for about 75% of the totalprotein mass of the vault particle. The two other proteins, the vaultpoly-ADP ribose polymerase, VPARP, a 193.3 kDa protein in humans, andthe telomerase/vault associated protein 1, TEP1, a 292 kDa protein inRattus norvegicus and a 290 kDa protein in humans, are each present inbetween about 2 and 16 copies per vault.

VPARP, is a poly ADP-ribosyl polymerase apparently unique to vaults. Itincludes a region of about 350 amino acids that shares 28% identity withthe catalytic domain of poly ADP-ribosyl polymerase, PARP, a nuclearprotein that catalyzes the formation of ADP-ribose polymers in responseto DNA damage. VPARP catalyzes an NAD-dependent poly ADP-ribosylationreaction, and purified vaults have poly ADP-ribosylation activity thattargets MVP, as well as VPARP itself.

Cryo-electron microscopy studies have determined that the vaults arehollow, barrel-like structures with two protruding end caps and aninvaginated waist. Regular small openings surround the vault cap. Theseopenings are large enough to allow small molecules and ions to enter theinterior of the vault. The volume of the internal cavity of the vault isabout 5×10⁷ Å³, large enough to enclose two ribosomes.

SUMMARY

According to one embodiment of the present invention, there is provideda method of using vaults as carrier molecules to deliver one or morethan one substance to an organism, or to a specific tissue or tospecific cells, or to an environmental medium. The method comprisesproviding vaults, incorporating the one or more than one substance intothe vaults, and administering the vaults comprising the one or more thanone substance to the organism, to the specific tissue, to the specificcells, or to the environmental medium. In one embodiment, the vaultsprovided are purified from natural sources. In another embodiment, thevaults provided are generated using recombinant technology. In oneembodiment, incorporation is accomplished by incubating the vaults withthe one or more than one substance. In one embodiment, the one or morethan one substance is selected from the group consisting of an enzyme, apharmaceutical agent, a plasmid, a polynucleotide, a polypeptide, asensor and a combination of the preceding.

According to another embodiment of the present invention, there isprovided a vault-like particle comprising MVP. In one embodiment, thevault-like particle further comprises VPARP or modified VPARP, or aportion of VPARP or a modified portion of VPARP. In another embodiment,the vault-like further comprises TEP1 or modified TEP1, or a portion ofTEP1 or a modified portion of TEP1.

According to another embodiment of the present invention, there isprovided a vault-like particle comprising modified MVP. In oneembodiment, the modified MVP comprises an amino acid sequence added tothe N-terminal of the MVP which results in one or more than onesubstance-binding domain within the vault-like particle. In anotherembodiment, the one or more than one substance-binding domain is between1 and 95 substance-binding domains. In another embodiment, the one ormore than one substance-binding domain is 96 substance-binding domains.In another embodiment, the one or more than one substance-binding domainis greater than 96 substance-binding domains. In one embodiment, the oneor more than one substance-binding domain within the vault-like particleis one or more than one heavy metal binding domain. In a preferredembodiment, the one or more than one heavy metal binding domain binds aheavy metal selected from the group consisting of cadmium, copper, goldand mercury. In a preferred embodiment, the peptide added to theN-terminal is a cysteine-rich peptide. In a preferred embodiment, theone or more than one substance-binding domain within the vault-likeparticle is one or more than one polynucleotide-binding domain. In apreferred embodiment, the one or more than one polynucleotide-bindingdomain is a non-specific polynucleotide-binding peptide. In a preferredembodiment, the one or more than one polynucleotide-binding domain is aspecific polynucleotide-binding peptide.

In another embodiment, the modified MVP of the vault-like particlecomprising modified MVP comprises an amino acid sequence added to theN-terminal of the MVP creates a sensor in the vault-like particle. Inone embodiment, the sensor is selected from the group consisting of achemical sensor, an ionic sensor, a microorganism sensor, an opticalsensor and a pH sensor. In one embodiment, the sensor is a greenfluorescent protein.

In another embodiment, the modified MVP of the vault-like particlecomprising modified MVP comprises an amino acid sequence added to theC-terminal of the MVP which results in one or more than onereceptor-binding domain. In one embodiment, the one or more than onereceptor-binding domain is between 1 and 95 receptor-binding domains. Inanother embodiment, the one or more than one receptor-binding domain is96 receptor-binding domains. In another embodiment, the one or more thanone receptor-binding domain is greater than 96 receptor-binding domains.In one embodiment, the one or more than one receptor-binding domain isnon-specific. In another embodiment, the one or more than onereceptor-binding domain is specific.

In another embodiment, the modified MVP further comprises an amino acidsequence added to the C-terminal of the MVP which results in one or morethan one receptor-binding domain. In one embodiment, the one or morethan one receptor-binding domain is between 1 and 95 receptor-bindingdomains. In another embodiment, the one or more than onereceptor-binding domain is 96 receptor-binding domains. In anotherembodiment, the one or more than one receptor-binding domain, is greaterthan 96 receptor-binding domains. In one embodiment, the one or morethan one receptor-binding domain is non-specific. In another embodiment,the one or more than one receptor-binding domain is specific.

In another embodiment, the modified MVP comprises both an amino acidsequence added to the C-terminal of the MVP and an amino acid sequenceadded to the N-terminal of the MVP.

According to another embodiment of the present invention, there isprovided a vault-like particle comprising MVP or modified MVP, andfurther comprises VPARP or a portion of VPARP comprising at least about150 consecutive residues of VPARP. In one embodiment, the portion ofVPARP comprises residues from about residue 1562 to 1724 of human VPARP,SEQ ID NO:3. In another embodiment, the portion of VPARP comprisesresidues from about residue 1473 to 1724 of human VPARP, SEQ ID NO:3. Inanother embodiment, the VPARP or portion of VPARP is modified. In oneembodiment, the modification comprises adding an amino acid sequenceadded to the C-terminal of the VPARP or portion of VPARP. In anotherembodiment, the modification comprises adding an amino acid sequenceadded to the N-terminal of the VPARP or portion of VPARP. In anotherembodiment, the modification comprises adding an amino acid sequenceadded to both the C-terminal and the N-terminal of the VPARP or portionof VPARP. In one embodiment, the modified MVP comprises an amino acidsequence added to the C-terminal of the MVP. In another embodiment, themodified MVP comprises an amino acid sequence added to the N-terminal ofthe MVP. In another embodiment, the modified MVP comprises both apeptide added to the C-terminal and a peptide added to the N-terminal.

According to another embodiment of the present invention, there isprovided a method of preventing damage by one or more than one substanceto an organism, to a specific tissue, to specific cells, or to anenvironmental medium, by sequestering the one or more than one substancewithin a vault-like particle. The method comprises providing vault-likeparticles, administering the vault-like particles to the organism,tissue, cells or environmental medium, and allowing the vault-likeparticles to sequester the one or more than one substance within thevault-like particles. In one embodiment, the one or more than onesubstance is a heavy metal selected from the group consisting ofcadmium, copper, gold and mercury. In another embodiment, the one ormore than one substance is a toxin selected from the group consisting ofarsenate, dioxin, an organochlorine, a pentachlorophenol and apolychlorinated biphenyl. In one embodiment, providing the vault-likeparticles comprises expressing the vault-like particles in a eukaryoticorganism.

According to another embodiment of the present invention, there isprovided a method of delivering one or more than one substance to anorganism, to a specific tissue, to specific cells, or to anenvironmental medium. The method comprises providing vault-likeparticles comprising the one or more than one substance, andadministering the vault-like particles comprising the one or more thanone substance to the organism, tissue, cells or environmental medium. Inone embodiment, the vault-like particles comprise, consist essentiallyof or consist of a modified MVP in addition to the one or more than onesubstance. In another embodiment, the vault-like particles comprise amodified VPARP or modified portion of VPARP. In another embodiment, thevault-like particles comprise both a modified MVP according to thepresent invention, and a modified VPARP or modified portion of VPARP. Inanother embodiment, the one or more than one substance is selected fromthe group consisting of an enzyme, a pharmaceutical agent, a plasmid, apolynucleotide, a polypeptide, a sensor and a combination of thepreceding. In another embodiment, the one or more than one substance isadenosine deaminase.

According to another embodiment of the present invention, there isprovided a method of delivering one or more than one sensor to anorganism, to a specific tissue, to specific cells, or to anenvironmental medium. The method comprises providing a vault-likeparticle comprising the one or more than one sensor and administeringthe vault-like particle to the organism, specific tissue, specificcells, or environmental medium. In one embodiment, the vault-likeparticles comprise, consist essentially of or consist of a modified MVP,in addition to the one or more than one sensor; In another embodiment,the vault-like particles comprise a modified VPARP or modified portionof VPARP. In another embodiment, the vault-like particles comprise botha modified MVP, and a modified VPARP or modified portion of VPARP. Inone embodiment, the sensor is selected from the group consisting of achemical sensor, a fluorescent sensor, an ionic sensor, a microorganismsensor, an optical sensor, and a pH sensor.

According to another embodiment of the present invention, there isprovided a method of detecting a signal from a sensor within anorganism, or a specific tissue or specific cells. The method comprisesdelivering one or more than one sensor to an organism, to a specifictissue, to specific cells, or to an environmental medium according tothe present invention, and detecting the presence of the sensor. In oneembodiment, detection is accomplished by fluorometry or byspectrophotometry.

According to another embodiment of the present invention, there isprovided a method of making vault-like particles. The method comprisescreating polynucleotide sequences encoding one or more than onepolypeptide selected from the group consisting of MVP, modified MVP,VPARP, a portion of VPARP, modified VPARP, a modified portion of VPARP,TEP1, a portion of TEP1, modified TEP1 and a modified portion of TEP1,using the polynucleotide sequences created to generate a bacmid DNA,using the bacmid DNA to generate a baculovirus comprising the sequence,and using the baculovirus to infect insect cells for protein productionusing an in situ assembly system.

According to another embodiment of the present invention, there isprovided a method of making vault-like particles comprising one or morethan one substance. The method comprises making vault-like particlesaccording to claim 63, and co-incubated the vault-like particles withthe one or more than one substance. In one embodiment, the one or morethan one substance is selected from the group consisting of enzyme, apharmaceutical agent, a plasmid, a polynucleotide, a polypeptide, asensor and a combination of the preceding. In another embodiment, themethod further comprises purifying the vault-like particles after makingthe vault-like particles.

DESCRIPTION

According to one embodiment of the present invention, there is provideda method of using vaults as carrier molecules to deliver one or morethan one substance to an organism, or to a specific tissue or specificcells. The method comprises administering vaults comprising thesubstance to the organism, tissue or cells.

According to another embodiment of the present invention, there isprovided a vault-like particle useful as a carrier molecule fordelivering one or more than one substance to a living system, such as anorganism, specific tissue or specific cell, or to an environmentalmedium. According to another embodiment of the present invention, thereis provided a method of delivering one or more than one substance to anorganism, or to a specific tissue or specific cells, or to anenvironmental medium. The method comprises providing vault-likeparticles comprising the substance, and administering the vault-likeparticles comprising the substance to the organism, tissue or cells, orto the environmental medium.

According to another embodiment of the present invention, there isprovided a method of delivering vault-like particles to a specifictissue or specific cells, or to an environmental medium. The methodcomprises providing vault-like particles having a receptor-bindingdomain on the surface of the vault-like particles, and administering thevault-like particles to the tissue or cells, or to the environmentalmedium.

According to another embodiment of the present invention, there isprovided a vault-like particle useful for sequestering the one or morethan one substance within the vault-like particle. According to anotherembodiment of the present invention, there is provided a method ofpreventing damage by one or more than one substance to an organism, orto a specific tissue or specific cells, or to an environmental medium,by sequestering the one or more than one substance within a vault-likeparticle. The method comprises providing vault-like particles comprisingone or more than one substance-binding domain within the vault-likeparticle, administering the vault-like particles to the organism, tissueor cells, or to the environmental medium, and allowing the vault-likeparticles to sequester the one or more than one substance within thevault-like particles.

Advantageously, both vaults and vault-like particles are resistant todegradation, such as intracellular degradation or environmentaldegradation, and therefore, can be used to deliver substances to or toremove substances from both living and non-living systems. Theembodiments of the present invention will now be disclosed in greaterdetail.

As used in this disclosure, “MVP,” “VPARP” and “TEP1” means the fullnaturally occurring polypeptide sequence. “vRNA” means the fullnaturally occurring polynucleotide sequence. As will be appreciated byone of ordinary skill in the art with reference to this disclosure, theactual sequence of any of MVP, VPARP, TEP1 and vRNAs can be from anyspecies suitable for the purposes disclosed in this disclosure, eventhough reference or examples are made to sequences from specificspecies. For example, when delivering substances to human organs ortissues, it is preferred to use human vaults or vault-like particlescomprising human sequences for MVP, VPARP, TEP1 and vRNAs. Further, aswill be appreciated by one of ordinary skill in the art with referenceto this disclosure, there are some intraspecies variations in thesequences of MVP, VPARP, TEP1 and vRNAs that are not relevant to thepurposes of the present invention. Therefore, references to MVP, VPARP,TEP1 and vRNAs are intended to include such intraspecies variants.

As used in this disclosure, the term “vault” or “vault particle,” ascompared to the term “vault-like particle” defined below, refers to anaturally occurring macro-molecular structure having MVP, VPARP, TEP1and one or more than one vRNA, whether purified from natural sources orgenerated through recombinant technology.

As used in this disclosure, the term “vault-like particle” refers to amacro-molecular structure comprising any of the following:

-   1) MVP without VPARP, TEP1 and vRNA;-   2) MVP and either VPARP or a portion of VPARP, without TEP1 and    vRNA;-   3) MVP and TEP1 or a portion of TEP1 with or without the one or more    than one vRNA, and without VPARP;-   4) MVP without VPARP, TEP1 and vRNA, where the MVP is modified to    attract a specific substance within the vault-like particle, or    modified to attract the vault-like particle to a specific tissue,    cell type or environmental medium, or modified both to attract a    specific substance within the vault-like particle and to attract the    vault particle to a specific tissue, cell type or environmental    medium; and-   5) MVP, and either VPARP or a portion of VPARP, or TEP1 or a portion    of TEP1 with or without the one or more than one vRNA, or with both    VPARP or a portion of VPARP, and TEP1, with or without the one or    more than one vRNA, where one or more than one of the MVP, VPARP or    portion of VPARP and TEP1 is modified to attract a specific    substance within the vault-like particle, or modified to attract the    vault particle to a specific tissue, cell type or environmental    medium, or modified both to attract a specific substance within the    vault-like particle and to attract the vault particle to a specific    tissue, cell type or environmental medium.

As used in this disclosure, the term “modified” and variations of theterm, such as “modification,” means one or more than one change to thenaturally occurring sequence of MVP, VPARP or TEP1 selected from thegroup consisting of addition of a polypeptide sequence to theC-terminal, addition of a polypeptide sequence to the N-terminal,deletion of between about 1 and 100 amino acid residues from theC-terminal, deletion of between about 1 and 100 amino acid residues fromthe N-terminal, substitution of one or more than one amino acid residuethat does not change the function of the polypeptide, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure, such as for example, an alanine to glycine substitution, anda combination of the preceding.

As used in this disclosure, the term “human” means “Homo sapiens.”

As used in this disclosure, the terms “organism,” “tissue” and “cell”include naturally occurring organisms, tissues and cells, geneticallymodified organisms, tissues and cells, and pathological tissues andcells, such as tumor cell lines in vitro and tumors in vivo.

As used in this disclosure, the term “environmental medium” means anon-living composition, composite, material, or mixture.

As used in this disclosure, the term “administering” includes anysuitable route of administration, as will be appreciated by one ofordinary skill in the art with reference to this disclosure, includingdirect injection into a solid organ, direct injection into a cell masssuch as a tumor, inhalation, intraperitoneal injection, intravenousinjection, topical application on a mucous membrane, or application toor dispersion within an environmental medium, and a combination of thepreceding. In one embodiment, the dosage of vaults or vault-likeparticles, with or without one or more than one substance enclosedwithin the vaults or vault-like particles, is between about 0.1 and10,000 micrograms per kilogram of body weight or environmental medium.In another embodiment, the dosage of vaults or vault-like particles,with or without one or more than one substance enclosed within thevaults or vault-like particles, is between about 1 and 1,000 microgramsper kilogram of body weight or environmental medium. In anotherembodiment, the dosage of vaults or vault-like particles, with orwithout one or more than one substance enclosed within the vaults orvault-like particles, is between about 10 and 1,000 micrograms perkilogram of body weight or environmental medium. For intravenousinjection and intraperitoneal injection, the dosage is preferablyadministered in a final volume of between about 0.1 and 10 ml. Forinhalation the dosage is preferably administered in a final volume ofbetween about 0.01 and 1 ml. As will be appreciated by one of ordinaryskill in the art with reference to this disclosure, the dose can berepeated a one or more than one of times as needed using the sameparameters to effect the purposes disclosed in this disclosure.

As used in this disclosure, “MS2” means the Enterobacteriophage MS2 coatprotein, which is an RNA-binding protein that specifically binds a 21-ntRNA stem-loop with high affinity.

As used in this disclosure, the term “comprise” and variations of theterm, such as “comprising” and “comprises,” are not intended to excludeother additives, components, integers or steps.

In one embodiment, the present invention is a method of using naturallyoccurring vaults as carrier molecules to deliver one or more than onesubstance to an organism, or to a specific tissue or specific cells, orto an environmental medium. The method comprises, first, providingvaults. In one embodiment, the vaults are purified from natural sources,such as mammalian liver or spleen tissue, using methods known to thosewith skill in the art, such as for example tissue homogenization,differential centrifugation, discontinuous sucrose gradientfractionation and cesium chloride gradient fractionation. In anotherembodiment, the vaults are made using recombinant technology. Next, theone or more than one substance is incorporated into the provided vaults.In a preferred embodiment, incorporation is accomplished by incubatingthe vaults with the one or more than one substance at an appropriatetemperature and for an appropriate time, as will be appreciated by oneof ordinary skill in the art with reference to this disclosure. Thevaults containing the one or more than one substance are then purified,such as for example sucrose gradient fractionation, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure. In a preferred embodiment, the one or more than onesubstance is selected from the group consisting of an enzyme, apharmaceutical agent, a plasmid, a polynucleotide, a polypeptide, asensor and a combination of the preceding. Next, the vaults comprisingthe one or more than one substance are administered to an organism, to aspecific tissue, to specific cells, or to an environmental medium.Administration is accomplished using any suitable route, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure.

According to another embodiment of the present invention, there isprovided a vault-like particle useful as a carrier molecule fordelivering one or more than one substance to an organism, to a specifictissue, to specific cells, or to an environmental medium, or useful forpreventing damage by one or more than one substance to an organism, to aspecific tissue, to specific cells, or to an environmental medium, bysequestering the one or more than one substance within a vault-likeparticle. The vault-like particle comprises MVP or modified MVP, and canfurther comprise VPARP or modified VPARP, a portion of VPARP or amodified portion of VPARP, and TEP1 or modified TEP1, a portion of TEP1or a modified portion of TEP1 with or without the one or more than onevRNA. In a preferred embodiment, the modifications are designed toattract a specific substance within the vault-like particle, to attractthe vault-like particle to a specific tissue or cell type, or both toattract a specific substance within the vault-like particle and toattract the vault particle to a specific tissue or cell type.

In one embodiment, the MVP is human MVP, SEQ ID NO:1, GenBank accessionnumber CAA56256, encoded by the cDNA, SEQ ID NO:2, GenBank accessionnumber X79882. In another embodiment, the VPARP is human VPARP, SEQ IDNO:3, GenBank accession number AAD47250, encoded by the cDNA, SEQ IDNO:4, GenBank accession number AF158255. In another embodiment, the TEP1is human TEP1, SEQ ID NO:5, GenBank accession number AAC51107, encodedby the cDNA, SEQ ID NO:6, GenBank accession number U86136. In anotherembodiment, the vRNA is human vRNA, SEQ ID NO:7, GenBank accessionnumber AF045143, SEQ ID NO:8, GenBank accession number AF045144, or SEQID NO:9, GenBank accession number AF045145, or a combination of thepreceding.

In one embodiment, the MVP is Rattus norvegicus MVP, SEQ ID NO:10,GenBank accession number AAC52161, encoded by the cDNA, SEQ ID NO:11,GenBank accession number U09870. In another embodiment, the TEP1 isRattus norvegicus TEP1, SEQ ID NO:12, GenBank accession number AAB51690,encoded by the cDNA, SEQ ID NO:13, GenBank accession number U89282. Inanother embodiment, the vRNA is Rattus norvegicus vRNA, SEQ ID NO:14,GenBank accession number Z1171. As can be seen, Rattus norvegicus MVPand human MVP share over 90% homology.

The following disclosure of vault protein modifications referencesspecific examples using specific human and Rattus norvegicus sequencesof MVP, VPARP and TEP1 sequences, however, as will be appreciated by oneof ordinary skill in the art with reference to this disclosure,corresponding modifications can be made using other sequences of thesespecies and can be made using sequences from other species asappropriate for the disclosed purposes.

According to one embodiment of the present invention, there is provideda vault-like particle comprising, consisting essentially of, orconsisting of modified MVP. In a preferred embodiment, the modificationcomprises adding an amino acid sequence to the N-terminal of the MVPwhich results in one or more than one substance-binding domain withinthe vault-like particle. When each copy of the MVP is modified in thismanner, one or more than one of the substance-binding domains, such as96 substance-binding domains, is present in each vault-like particle,however, vault-like particles can also be assembled from a mixture ofMVP with the N-terminal modified and MVP without the N-terminalmodified, to create vault-like particle with less than 96substance-binding domains in the vault-like particle, and the addedamino acid terminal sequences can be polymerized as will be appreciatedby one of ordinary skill in the art with reference to this disclosure tocreate more than 96 substance-binding domains in the vault-likeparticle.

In a preferred embodiment, there is provided a vault-like particlecomprising, consisting essentially of, or consisting of an MVP modifiedby adding a peptide to the N-terminal to create a one or more than oneof heavy metal binding domains. In a preferred embodiment, the heavymetal binding domains bind a heavy metal selected from the groupconsisting of cadmium, copper, gold and mercury. In a preferredembodiment, the peptide added to the N-terminal is a cysteine-richpeptide (CP), such as for example, SEQ ID NO:15, the MVP is human MVP,SEQ ID NO:1, and the modification results in CP-MVP, SEQ ID NO:16,encoded by the cDNA, SEQ ID NO:17. In another preferred embodiment, thecysteine-rich peptide is SEQ ID NO:15, the MVP is Rattus norvegicus MVP,SEQ ID NO:10, and the modification results in CP-MVP, SEQ ID NO:18,encoded by the cDNA, SEQ ID NO:19. These embodiments are particularlyuseful because vault-like particles consisting of CP-MVP, SEQ ID NO:16or SEQ ID NO:18, are stable without the presence of other vaultproteins.

In another embodiment, there is provided a vault-like particlecomprising, consisting essentially of, or consisting of an MVP modifiedby adding a peptide to the N-terminal to create one or more than onepolynucleotide-binding domain. In a preferred embodiment, the peptide isa non-specific polynucleotide-binding peptide, such as for example,HisT7, SEQ ID NO:20, encoded by the cDNA, SEQ ID NO:21, or a polylysinesuch as SEQ ID NO:22, encoded by the cDNA, SEQ ID NO:23, the MVP ishuman MVP, SEQ ID NO:1, and the modification results in HisT7-MVP, SEQID NO:24, encoded by the cDNA, SEQ ID NO:25, or in polylysine-MVP, SEQID NO:26, encoded by the cDNA, SEQ ID NO:27, respectfully. In anotherpreferred embodiment, the peptide is a non-specificpolynucleotide-binding peptide, such as for example, HisT7, SEQ IDNO:20, encoded by the cDNA, SEQ ID NO:21, or a polylysine such as SEQ IDNO:22, encoded by the cDNA, SEQ ID NO:23, the MVP is Rattus norvegicusMVP, SEQ ID NO:10, and the modification results in HisT7-MVP, SEQ IDNO:28, encoded by the cDNA, SEQ ID NO:29, or in polylysine-MVP, SEQ IDNO:30, encoded by the cDNA, SEQ ID NO:31, respectfully. HisT7-MVP, SEQID NO:24 and SEQ ID NO:28, are examples of modified MVP that can also beused to bind specific antibodies within the vault-like particle, inthese cases, the T7 monoclonal antibody, but corresponding modificationscan be made to bind other specific antibodies, as will be appreciated byone of ordinary skill in the art with reference to this disclosure. Inanother preferred embodiment, the peptide is a specific DNA bindingpeptide, such as for example, GAL4, SEQ ID NO:32, encoded by the cDNA,SEQ ID NO:33, the MVP is human MVP, SEQ ID NO:1, and the modificationresults in GAL4-MVP, SEQ ID NO:34, encoded by the cDNA, SEQ ID NO:35. Inanother preferred embodiment, the peptide is a specific DNA bindingpeptide, such as for example, GAL4, SEQ ID NO:32, encoded by the cDNA,SEQ ID NO:33, the MVP is Rattus norvegicus MVP, SEQ ID NO:10, and themodification results in GAL4-MVP, SEQ ID NO:36, encoded by the cDNA, SEQID NO:37. In another preferred embodiment, the peptide is a specific RNAbinding peptide, such as for example, MS2, SEQ ID NO:38, encoded by thecDNA, SEQ ID NO:39, the MVP is human MVP, SEQ ID NO:1, and themodification results in MS2-MVP, SEQ ID NO:40, encoded by the cDNA, SEQID NO:41. In another preferred embodiment, the peptide is an RNA bindingpeptide, such as for example, MS2, SEQ ID NO:38, encoded by the cDNA,SEQ ID NO:39, the MVP is Rattus norvegicus MVP, SEQ ID NO:10, and themodification results in MS2-MVP, SEQ ID NO:42, encoded by the cDNA, SEQID NO:43.

In another embodiment, there is provided a vault-like particlecomprising, consisting essentially of, or consisting of an MVP modifiedby adding a peptide to the N-terminal to create a sensor in thevault-like particle. The sensor can be any suitable sensor, as will beappreciated by one of ordinary skill in the art with reference to thisdisclosure, such as for example, a chemical sensor such as a cyclic-AMPbinding protein, an ionic sensor such as a calcium or potassium sensor,a microorganism sensor such an antibody specific for E. coli, an opticalsensor such as a quantum dot, and a pH sensor such as green fluorescenceprotein. In a preferred embodiment, the sensor is a fluorescent protein,such as green fluorescent protein (GL), SEQ ID NO:44, encoded by thecDNA, SEQ ID NO:45, the MVP is human MVP, SEQ ID NO:1, and themodification results in GL-MVP, SEQ ID NO:46, encoded by the cDNA, SEQID NO:47. In another preferred embodiment, the sensor is a fluorescentprotein, such as green fluorescent protein (GL), SEQ ID NO:44, encodedby the cDNA, SEQ ID NO:45, the MVP is Rattus norvegicus MVP, SEQ IDNO:10, and the modification results in GL-MVP, SEQ ID NO:48, encoded bythe cDNA, SEQ ID NO:49.

In another embodiment, there is provided a vault-like particlecomprising MVP or modified MVP, and further comprising VPARP or aportion of VPARP comprising at least about 150 consecutive residues ofVPARP, and modified by adding a peptide to either the C-terminal or theN-terminal to create a one or more than one of substance-binding domainsor a one or more than one of sensors within the vault-like particleshaving the same purposes as disclosed with reference to modified MVP inthis disclosure. By way of example only, in one embodiment, the residuesare from about residue 1562 to residue 1724 of human VPARP, SEQ ID NO:3.In another embodiment, the residues are from about residue 1473 toresidue 1724 of human VPARP, SEQ ID NO:3. The substance-binding domainson the VPARP or portion of VPARP serve the same functions as disclosedin this disclosure with respect to N-terminal modifications of MVP. Forexample, in one embodiment, the vault-like particles comprise residues1473-1724 of VPARP, SEQ ID NO:3, modified by adding CP, SEQ ID NO:15, tothe N-terminal, to create (1473-1724)CP-VPARP, SEQ ID NO:50, encoded bythe cDNA, SEQ ID NO:51. In another embodiment, the vault-like particlescomprise VPARP, SEQ ID NO:3, modified by adding CP, SEQ ID NO:15, to theN-terminal, to create CP-VPARP, SEQ ID NO:52, encoded by the cDNA, SEQID NO:53. In one embodiment, the vault-like particles comprise residues1473-1724 of VPARP, SEQ ID NO:3, modified by adding GAL4, SEQ ID NO:32,to the N-terminal, to create GAL4-(1473-1724)VPARP, SEQ ID NO:54,encoded by the cDNA, SEQ ID NO:55. In another embodiment, the vault-likeparticles comprise VPARP, SEQ ID NO:3, modified by adding GAL4, SEQ IDNO:32, to the N-terminal, to create GAL4-VPARP, SEQ ID NO:56, encoded bythe cDNA, SEQ ID NO:57. In another embodiment, the vault-like particlescomprise residues 1473-1724 of VPARP, SEQ ID NO:3, modified by addingGL, SEQ ID NO:44, to the N-terminal, to create GL-(1473-1724)VPARP, SEQID NO:58, encoded by the cDNA, SEQ ID NO:59. In another embodiment, thevault-like particles comprise VPARP, SEQ ID NO:3, modified by adding GL,SEQ ID NO:44, to the N-terminal, to create GL-VPARP, SEQ ID NO:60,encoded by the cDNA, SEQ ID NO:61. In another embodiment, the vault-likeparticles comprise residues 1473-1724 of VPARP, SEQ ID NO:3, modified byadding MS2, SEQ ID NO:38, to the N-terminal, to createMS2-(1473-1724)VPARP, SEQ ID NO:62, encoded by the cDNA, SEQ ID NO:63.In another embodiment, the vault-like particles comprise VPARP, SEQ IDNO:3, modified by adding MS2, SEQ ID NO:38, to the N-terminal, to createMS2-VPARP, SEQ ID NO:64, encoded by the cDNA, SEQ ID NO:65. In anotherembodiment, the vault-like particles comprise residues 1473-1724 ofVPARP, SEQ ID NO:3, modified by adding a Photinus pyralis luciferase(LUC), SEQ ID NO:66 GenBank accession number P08659, encoded by thepGL3-Basic vector SEQ ID NO:67, GenBank accession number U47295 to theN-terminal, to create LUC-(1473-1724)VPARP, SEQ ID NO:68, encoded by thecDNA, SEQ ID NO:69. In another embodiment, the vault-like particlescomprise VPARP, SEQ ID NO:3, modified by adding LUC, SEQ ID NO:66, tothe N-terminal, to create LUC-VPARP, SEQ ID NO:71, encoded by the cDNA,SEQ ID NO:72. Further, as will be appreciated by one of ordinary skillin the art with reference to this disclosure, the present invention alsoincludes corresponding modifications to the C-terminal of VPARP or aportion of VPARP, and serve the same function. In a preferredembodiment, the substance binding domain binds the enzyme adenosinedeaminase.

According to one embodiment of the present invention, there is provideda vault-like particle comprising, consisting essentially of, orconsisting of MVP modified by adding an amino acid sequence to theC-terminal of the MVP which results in one or more than onereceptor-binding domain, such as a protein targeting domain, on thesurface of the vault-like particle. When each copy of the MVP ismodified in this manner, one or more than one of the receptor-bindingdomains, such as 96 receptor-binding domains, is present on eachvault-like particle, however, vault-like particles can also be assembledfrom a mixture of MVP with the C-terminal modified and MVP without theC-terminal modified, to create vault-like particle with less than 96receptor-binding domains on the vault-like particle.

In a preferred embodiment, there is provided a vault-like particlecomprising, consisting essentially of, or consisting of an MVP modifiedby adding a peptide to the C-terminal to create a one or more than oneof eukaryotic cell receptor-binding domains on the exterior of thevault-like particles. In a preferred embodiment, the eukaryotic cellreceptor-binding domain is generally non-specific. For example, in oneembodiment, the peptide is Antennapedia (ANT), SEQ ID NO:72, encoded bythe cDNA, SEQ ID NO:73, the MVP is human MVP, SEQ ID NO:1, and themodification results in MVP-ANT, SEQ ID NO:74, encoded by the cDNA, SEQID NO:75. In another embodiment, the peptide is ANT, SEQ ID NO:72,encoded by the cDNA, SEQ ID NO:73, the MVP is Rattus norvegicus MVP, SEQID NO:10, and the modification results in MVP-ANT, SEQ ID NO:76, encodedby the cDNA, SEQ ID NO:77. In another embodiment, the peptide is HIV-Tat(TAT), SEQ ID NO:78, encoded by the cDNA, SEQ ID NO:79, the MVP is humanMVP, SEQ ID NO:1, and the modification results in MVP-TAT, SEQ ID NO:80,encoded by the cDNA, SEQ ID NO:81. In another embodiment, the peptide isTAT, SEQ ID NO:78, encoded by the cDNA, SEQ ID NO:79, the MVP is Rattusnorvegicus MVP, SEQ ID NO:10, and the modification results in MVP-TAT,SEQ ID NO:82, encoded by the cDNA, SEQ ID NO:83. In another embodiment,the eukaryotic cell receptor-binding domain is specific to a certaintype of eukaryotic cell receptor, such as for example a carcinoembryonicantigen receptor, a protein found on the surface of about 50% of allhuman tumors, or an epidermal growth factor (EGF) receptor. For example,in one embodiment, the peptide is anti-CEA scFv diabody (αCEA), SEQ IDNO:84, encoded by the cDNA, SEQ ID NO:85, the MVP is human MVP, SEQ IDNO:1, and the modification results in MVP-αCEA, SEQ ID NO:86, encoded bythe cDNA, SEQ ID NO:87. In another embodiment, the peptide is αCEA, SEQID NO:84, encoded by the cDNA, SEQ ID NO:85, the MVP is Rattusnorvegicus MVP, SEQ ID NO:10, and the modification results in MVP-αCEA,SEQ ID NO:88, encoded by the cDNA, SEQ ID NO:89. In another embodiment,the peptide is EGF, SEQ ID NO:90, encoded by the cDNA, SEQ ID NO:91, theMVP is human MVP, SEQ ID NO:1, and the modification results in MVP-EGF,SEQ ID NO:92, encoded by the cDNA, SEQ ID NO:93. In another embodiment,the peptide is EGF, SEQ ID NO:90, encoded by the cDNA, SEQ ID NO:91, theMVP is Rattus norvegicus MVP, SEQ ID NO:10, and the modification resultsin MVP-EGF, SEQ ID NO:94, encoded by the cDNA, SEQ ID NO:95.

According to one embodiment of the present invention, there is provideda vault-like particle comprising, consisting essentially of, orconsisting of MVP modified by adding an amino acid sequence to theN-terminal and also modified by adding an amino acid sequence to theC-terminal. The modification of the N-terminal and the modification ofthe C-terminal can be any modification as disclosed in this disclosure,for the same purposes as disclosed in this disclosure. For example, themodification of the N-terminal can result in a substance-binding domain,such as for example a heavy metal binding domain or a polynucleotidebinding domain, or can result in a sensor within the vault-likeparticle. The modification of the C-terminal can result in one or morethan one receptor-binding domain on the surface of the vault-likeparticle. By way of example only, in one embodiment, the vault-likeparticle comprises, consists essentially of, or consists of MVP modifiedby adding GAL4, SEQ ID NO:32, to the N-terminal of human MVP, SEQ IDNO:1, and ANT, SEQ ID NO:72 to the C-terminal of human MVP, SEQ ID NO:1,to create GAL4-MVP-ANT, SEQ ID NO:96, encoded by the cDNA, SEQ ID NO:97.In another embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding GAL4, SEQ IDNO:32, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, andANT, SEQ ID NO:72 to the C-terminal of Rattus norvegicus MVP, SEQ IDNO:10, to create GAL4-MVP-ANT, SEQ ID NO:98, encoded by the cDNA, SEQ IDNO:99. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by adding GAL4, SEQID NO:32, to the N-terminal of human MVP, SEQ ID NO:1, and αCEA, SEQ IDNO:84 to the C-terminal of human MVP, SEQ ID NO:1, to createGAL4-MVP-αCEA, SEQ ID NO:100, encoded by the cDNA, SEQ ID NO:101. Inanother embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding GAL4, SEQ IDNO:32, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, andαCEA, SEQ ID NO:84 to the C-terminal of Rattus norvegicus MVP, SEQ IDNO:10, to create GAL4-MVP-αCEA, SEQ ID NO:102, encoded by the cDNA, SEQID NO:103. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by adding GAL4, SEQID NO:32, to the N-terminal of human MVP, SEQ ID NO:1, and EGF, SEQ IDNO:90 to the C-terminal of human MVP, SEQ ID NO:1, to createGAL4-MVP-EGF, SEQ ID NO:104, encoded by the cDNA, SEQ ID NO:105. Inanother embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding GAL4, SEQ IDNO:32, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, andEGF, SEQ ID NO:90 to the C-terminal of Rattus norvegicus MVP, SEQ IDNO:10, to create GAL4-MVP-EGF, SEQ ID NO:106, encoded by the cDNA, SEQID NO:107. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by adding GAL4, SEQID NO:32, to the N-terminal of human MVP, SEQ ID NO:1, and TAT, SEQ IDNO:78 to the C-terminal of human MVP, SEQ ID NO:1, to createGAL4-MVP-TAT, SEQ ID NO:108, encoded by the cDNA, SEQ ID NO:109. Inanother embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding GAL4, SEQ IDNO:32, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:1, and TAT,SEQ ID NO:78 to the C-terminal of Rattus norvegicus MVP, SEQ ID NO:10,to create GAL4-MVP-TAT, SEQ ID NO:110, encoded by the cDNA, SEQ IDNO:111. In one embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding MS2, SEQ ID NO:38,to the N-terminal of human MVP, SEQ ID NO:1, and ANT, SEQ ID NO:72 tothe C-terminal of human MVP, SEQ ID NO:1, to create MS2-MVP-ANT, SEQ IDNO:112, encoded by the cDNA, SEQ ID NO:113. In another embodiment, thevault-like particle comprises, consists essentially of, or consists ofMVP modified by adding MS2, SEQ ID NO:38, to the N-terminal of Rattusnorvegicus MVP, SEQ ID NO:10, and ANT, SEQ ID NO:72 to the C-terminal ofRattus norvegicus MVP, SEQ ID NO:10, to create MS2-MVP-ANT, SEQ IDNO:114, encoded by the cDNA, SEQ ID NO:115. In another embodiment, thevault-like particle comprises, consists essentially of, or consists ofMVP modified by adding MS2, SEQ ID NO:38, to the N-terminal of humanMVP, SEQ ID NO:1, and αCEA, SEQ ID NO:84 to the C-terminal of human MVP,SEQ ID NO:1, to create MS2-MVP-αCEA, SEQ ID NO:116, encoded by the cDNA,SEQ ID NO:117. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by adding MS2, SEQID NO:38, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, andαCEA, SEQ ID NO:84 to the C-terminal of Rattus norvegicus MVP, SEQ IDNO:10, to create MS2-MVP-αCEA, SEQ ID NO:118, encoded by the cDNA, SEQID NO:119. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by adding MS2, SEQID NO:38, to the N-terminal of human MVP, SEQ ID NO:1, and EGF, SEQ IDNO:90 to the C-terminal of human MVP, SEQ ID NO:1, to createMS2-MVP-EGF, SEQ ID NO:120, encoded by the cDNA, SEQ ID NO:121. Inanother embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding MS2, SEQ ID NO:38,to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, and EGF, SEQID NO:90 to the C-terminal of Rattus norvegicus MVP, SEQ ID NO:10, tocreate MS2-MVP-EGF, SEQ ID NO:122, encoded by the cDNA, SEQ ID NO:123.In another embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding MS2, SEQ ID NO:38,to the N-terminal of human MVP, SEQ ID NO:1, and TAT, SEQ ID NO:78 tothe C-terminal of human MVP, SEQ ID NO:1, to create MS2-MVP-TAT, SEQ IDNO:124, encoded by the cDNA, SEQ ID NO:125. In another embodiment, thevault-like particle comprises, consists essentially of, or consists ofMVP modified by adding MS2, SEQ ID NO:38, to the N-terminal of Rattusnorvegicus MVP, SEQ ID NO:1, and TAT, SEQ ID NO:78 to the C-terminal ofRattus norvegicus MVP, SEQ ID NO:10, to create MS2-MVP-TAT, SEQ IDNO:126, encoded by the cDNA, SEQ ID NO:127. In one embodiment, thevault-like particle comprises, consists essentially of, or consists ofMVP modified by adding polylysine, SEQ ID NO:22, to the N-terminal ofhuman MVP, SEQ ID NO:1, and ANT, SEQ ID NO:72 to the C-terminal of humanMVP, SEQ ID NO:1, to create polylysine-MVP-ANT, SEQ ID NO:128, encodedby the cDNA, SEQ ID NO:129. In another embodiment, the vault-likeparticle comprises, consists essentially of, or consists of MVP modifiedby adding polylysine, SEQ ID NO:22, to the N-terminal of Rattusnorvegicus MVP, SEQ ID NO:10, and ANT, SEQ ID NO:72 to the C-terminal ofRattus norvegicus MVP, SEQ ID NO:10, to create polylysine-MVP-ANT, SEQID NO:130, encoded by the cDNA, SEQ ID NO:131. In another embodiment,the vault-like particle comprises, consists essentially of, or consistsof MVP modified by adding polylysine, SEQ ID NO:22, to the N-terminal ofhuman MVP, SEQ ID NO:1, and αCEA, SEQ ID NO:84 to the C-terminal ofhuman MVP, SEQ ID NO:1, to create polylysine-MVP-αCEA, SEQ ID NO:132,encoded by the cDNA, SEQ ID NO:133. In another embodiment, thevault-like particle comprises, consists essentially of, or consists ofMVP modified by adding polylysine, SEQ ID NO:22, to the N-terminal ofRattus norvegicus MVP, SEQ ID NO:10, and αCEA, SEQ ID NO:84 to theC-terminal of Rattus norvegicus MVP, SEQ ID NO:10, to createpolylysine-MVP-αCEA, SEQ ID NO:134, encoded by the cDNA, SEQ ID NO:135.In another embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding polylysine, SEQ IDNO:22, to the N-terminal of human MVP, SEQ ID NO:1, and EGF, SEQ IDNO:90 to the C-terminal of human MVP, SEQ ID NO:1, to createpolylysine-MVP-EGF, SEQ ID NO:136, encoded by the cDNA, SEQ ID NO:137.In another embodiment, the vault-like particle comprises, consistsessentially of, or consists of MVP modified by adding polylysine, SEQ IDNO:22, to the N-terminal of Rattus norvegicus MVP, SEQ ID NO:10, andEGF, SEQ ID NO:90 to the C-terminal of Rattus norvegicus MVP, SEQ IDNO:10, to create polylysine-MVP-EGF, SEQ ID NO:138, encoded by the cDNA,SEQ ID NO:139. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by addingpolylysine, SEQ ID NO:22, to the N-terminal of human MVP, SEQ ID NO:1,and TAT, SEQ ID NO:78 to the C-terminal of human MVP, SEQ ID NO:1, tocreate polylysine-MVP-TAT, SEQ ID NO:140, encoded by the cDNA, SEQ IDNO:141. In another embodiment, the vault-like particle comprises,consists essentially of, or consists of MVP modified by addingpolylysine, SEQ ID NO:22, to the N-terminal of Rattus norvegicus MVP,SEQ ID NO:1, and TAT, SEQ ID NO:78 to the C-terminal of Rattusnorvegicus MVP, SEQ ID NO:10, to create polylysine-MVP-TAT, SEQ IDNO:142, encoded by the cDNA, SEQ ID NO:143.

According to another embodiment of the present invention, there isprovided a vault-like particle comprising MVP and VPARP or a portion ofVPARP, where the MVP is modified by adding an amino acid sequence to theN-terminal or is modified by adding an amino acid sequence to theC-terminal, or is modified both by adding an amino acid sequence to theN-terminal and by adding an amino acid sequence to the C-terminal, andwhere the VPARP or portion of VPARP is modified by adding an amino acidsequence to the N-terminal or is modified by adding an amino acidsequence to the C-terminal, or is modified both by adding an amino acidsequence to the N-terminal and by adding an amino acid sequence to theC-terminal. The modifications can be any modification as disclosed inthis disclosure, for the same purposes as disclosed in this disclosure.

In another embodiment of the present invention, there is provided amethod of preventing damage by one or more than one substance to anorganism, to a specific tissue, to specific cells, or to anenvironmental medium, by sequestering the one or more than one substancewithin a vault-like particle. The method comprises providing vault-likeparticles according to the present invention. The method furthercomprises administering the vault-like particles to the organism,tissue, cells or environmental medium, and allowing the vault-likeparticles to sequester the one or more than one substance within thevault-like particles. In one embodiment, the vault-like particlescomprise, consist essentially of or consist of a modified MVP accordingto the present invention. In another embodiment, the vault-likeparticles comprise a modified VPARP or portion of VPARP according to thepresent invention. In another embodiment, the vault-like particlescomprise both a modified MVP according to the present invention, and amodified VPARP or portion of VPARP according to the present invention.In a preferred embodiment, the vault-like particles comprise, consistessentially of or consist of MVP modified by adding a peptide to theN-terminal to create a one or more than one of heavy metal bindingdomains. In one embodiment, the one or more than one substance is aheavy metal selected from the group consisting of cadmium, copper, goldand mercury. In another embodiment, the one or more than one substanceis a toxin selected from the group consisting of arsenate, dioxin, anorganochlorine, a pentachlorophenol and a polychlorinated biphenyl. In apreferred embodiment, the providing step comprises expressing thevault-like particles in a eukaryotic organisms, such as for example anAcanthomoeba sp., yeast or Dictostelium discoidieum, capable ofproliferating in contaminated soil, and the administering step comprisesintroducing the organisms with the expressed vault-like particles intothe contaminated soil. For example, vault-like particles comprising anarsenate reductase enzyme within the vault-like particles can beexpressed in the organisms and used to detoxify soil. For example, inone embodiment, modified MVP is provided comprising one or more than onearsenate-binding domain at the N-terminal. Arsenate reductase enzyme iscloned with residues 1473-1724 of human VPARP, SEQ ID NO:3 at either theC-terminal or the N-terminal. Both proteins are co-expressed in aprimitive eukaryotic organisms, such as acanthomoeba, yeast orDictostelium discoidieum, capable of proliferating in contaminated soil.The organisms engineered to contain the two modified proteins areintroduced into contaminated soil, where they are exposed to theenvironmental toxin, such as arsenate. The expressed vault-likeparticles, comprising 96 or more copies of the arsenate-binding domainand the detoxification enzyme, arsenate reductase within the vault-likeparticles, then sequester and detoxify the environmental toxin, arsenatein the environmental medium.

In another embodiment of the present invention, there is provided amethod of delivering one or more than one substance to an organism, to aspecific tissue, to specific cells, or to an environmental medium. Themethod comprises providing vault-like particles according to the presentinvention comprising the one or more than one substance. The methodfurther comprises administering the vault-like particles comprising theone or more than one substance to the organism, tissue, cells orenvironmental medium. In one embodiment, the vault-like particlescomprise, consist essentially of or consist of a modified MVP accordingto the present invention, in addition to the one or more than onesubstance. In another embodiment, the vault-like particles comprise amodified VPARP or modified portion of VPARP according to the presentinvention. In another embodiment, the vault-like particles comprise botha modified MVP according to the present invention, and a modified VPARPor modified portion of VPARP according to the present invention. In apreferred embodiment, the one or more than one substance is selectedfrom the group consisting of an enzyme, a pharmaceutical agent, aplasmid, a polynucleotide, a polypeptide, a sensor and a combination ofthe preceding. In a particularly preferred embodiment, the substance isadenosine deaminase.

In another embodiment of the present invention, there is provided amethod of delivering one or more than one sensor to an organism, to aspecific tissue, to specific cells, or to an environmental medium. Themethod comprises providing a vault-like particle comprising the one ormore than one sensor and administering the vault-like particle to theorganism, specific tissue, specific cells, or environmental medium. Inone embodiment, the vault-like particles comprise, consist essentiallyof or consist of a modified MVP according to the present invention, inaddition to the one or more than one sensor. In another embodiment, thevault-like particles comprise a modified VPARP or modified portion ofVPARP according to the present invention. In another embodiment, thevault-like particles comprise both a modified MVP according to thepresent invention, and a modified VPARP or modified portion of VPARPaccording to the present invention. The sensor can be any suitablesensor, as will be appreciated by one of ordinary skill in the art withreference to this disclosure, such as for example, a chemical sensorsuch as a cyclic-AMP binding protein, an ionic sensor such as a calciumor potassium sensor, a microorganism sensor such an antibody specificfor E. coli, an optical sensor such as a quantum dot, and a pH sensorsuch as green fluorescence protein. In a preferred embodiment, thesensor is a fluorescent sensor.

In another embodiment, the present invention is a method of detecting asignal from a sensor within an organism, or a specific tissue orspecific cells. The method comprises delivering one or more than onesensor to an organism, to a specific tissue, to specific cells, or to anenvironmental medium, according to a method of the present invention.Then, the presence of the sensor is detected. Detection is performedusing standard techniques, such as for example, fluorometry orspectrophotometry. This method can be used, for example, to determinethe pH within cells, where the sensor is a pH dependent fluorescentsensor, as will be appreciated by one of ordinary skill in the art withreference to this disclosure.

According to another embodiment of the present invention, there isprovided a method of making vault-like particles according to thepresent invention. The method comprises creating polynucleotidesequences encoding one or more than one polypeptide selected from thegroup consisting of MVP, modified MVP, VPARP, a portion of VPARP,modified VPARP, a modified portion of VPARP, TEP1, a portion of TEP1,modified TEP1 and a modified portion of TEP1, using standard molecularbiological procedures, such as polymerase chain reaction and specificoligonucleotides, as will be appreciated by one of ordinary skill in theart with reference to this disclosure. Preferably, the polynucleotidesequences are used to generate a bacmid DNA that is used to generate abaculovirus comprising the sequence. The baculovirus is then used toinfect insect cells for protein production using an in situ assemblysystem, such as the baculovirus protein expression system, according tostandard techniques, as will be appreciated by one of ordinary skill inthe art with reference to this disclosure. Advantageously, we have usedthe baculovirus protein expression system to produce milligramquantities of vault-like particles, and this system can be scaled up toallow production of gram quantities of vault-like particles according tothe present invention.

In another embodiment of the present invention, there is provided amethod of making vault-like particles having one or more than onesubstance, such as an enzyme, a pharmaceutical agent, a plasmid, apolynucleotide, a polypeptide, a sensor and a combination of thepreceding, within the vault-like particles. The method comprises makingthe vault-like particles according to a method of the present invention.Next, the vault-like particles are purified using, such as for example,standard procedures over sucrose gradients. Then, the vault-likeparticles are co-incubated with one or more than one substance, untilthe one or more than one substance equilibrates within the vault-likeparticles or until enough of the one or more than one substance isloaded in the vault-like particles for the intended purpose.

Although the present invention has been discussed in considerable detailwith reference to certain preferred embodiments, other embodiments arepossible. Therefore, the scope of the appended claims should not belimited to the description of preferred embodiments contained in thisdisclosure. All references cited herein are incorporated by reference totheir entirety.

1. A method of delivering one or more than one substance, comprising: a)providing vault-like particles comprising a polypeptide, the polypeptidebeing linked to MVP, VPARP, a portion of VPARP, or a modified MVP,VPARP, or portion of VPARP; and b) administering the vault-likeparticles, wherein the substance or more than one substance comprisesthe polypeptide, an agent that binds preferentially to the polypeptide,or a combination thereof.
 2. The method of claim 1, where the one ormore than one substance is selected from the group consisting of anenzyme, a pharmaceutical agent, a plasmid, a polynucleotide, apolypeptide, a sensor and a combination of the preceding.
 3. The methodof claim 1, where the one or more than one substance is adenosinedeaminase.
 4. The method of claim 1, where the substance or more thanone substance is a sensor selected from the group consisting of achemical sensor, a fluorescent sensor, an ionic sensor, a microorganismsensor, an optical sensor, and a pH sensor.